Methods and compositions for delivering interleukin-1 receptor antagonist

ABSTRACT

Methods and compositions generating and using an interleukin-1 receptor antagonist (IL-1ra)-rich solution. Methods for generating and isolating interleukin-1 receptor antagonist include incubating adipose tissue and/or adipocytes with polyacrylamide beads to produce interleukin-1 receptor antagonist. The interleukin-1 receptor antagonist is isolated from the polyacrylamide beads to obtain the solution rich in interleukin-1 receptor antagonist. Methods for treating a site of inflammation in a patient include administering to the site of inflammation the solution rich in interleukin-1 receptor antagonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 12/394,723 filed on Feb. 27, 2009 which claims the benefit ofU.S. Provisional Application No. 61/031,803, filed on Feb. 27, 2008;U.S. Provisional Application No. 61/116,940, filed on Nov. 21, 2008; andU.S. Provisional Application No. 61/155,048, filed on Feb. 24, 2009. Theentire disclosures of each of the above applications are incorporatedherein by reference.

INTRODUCTION

The present technology relates to compositions comprising interleukin-1receptor antagonist, and methods for generating, isolating, anddelivering such compositions.

Interleukin-1 (IL-1) includes a family of cytokines that can stimulatelymphocytes and macrophages, activate phagocytes, increase prostaglandinproduction, contribute to degeneration of bone joints, increase bonemarrow cell proliferation, and are involved in many chronic inflammatoryconditions. IL-1 can be generated by macrophages, monocytes, anddendritic cells, and can be part of the inflammatory response againstinfection.

The mode of action of IL-1 can be mediated by interleukin-1 receptorantagonist protein (IL-1ra; also known as “IRAP”). IL-1ra binds to thesame receptor on the cell surface as IL-1, and thus prevents IL-1 fromsending a signal to that cell. IL-1ra is secreted from white bloodcells, including monocytes, macrophages, neutrophils, polymorphonuclearcells (PMNs), and other cells, and can modulate a variety of IL-1related immune and inflammatory responses, as described by Arend W P,Malyak M, Guthridge C J, Gabay C (1998) “Interleukin-1 receptorantagonist: role in biology” Annu. Rev. Immunol. 16: 27-55. Productionof IL-1ra is stimulated by several substances including adherentimmunoglobulin G (IgG), other cytokines, and bacterial or viralcomponents. IL-1ra is an important natural anti-inflammatory protein inarthritis, colitis, and granulomatous pulmonary disease.

IL-1ra can be used in the treatment of rheumatoid arthritis, anautoimmune disease in which IL-1 plays a key role, reducing inflammationand cartilage degradation associated with the disease. For example,Kineret™ (anakinra) is a recombinant, non-glycosylated form of IL-1ra(Amgen Manufacturing, Ltd., Thousand Oaks, Calif.). Various recombinantinterleukin-1 inhibitors and methods of treatment are described in U.S.Pat. No. 6,599,873, Sommer et al., issued Jul. 29, 2003; U.S. Pat. No.5,075,222, Hannum et al., issued Dec. 24, 1991; and U.S. ApplicationPublication No. 2005/0197293, Mellis et al., published Sep. 8, 2005 Inaddition, methods for producing IL-1ra from body fluids, including theuse of autologous fluids, are described in U.S. Pat. No. 6,623,472,Reincke et al., issued Sep. 23, 2003; U.S. Pat. No. 6,713,246, Reineckeet al., issued Mar. 30, 2004; and U.S. Pat. No. 6,759,188, Reinecke etal., issued Jul. 6, 2004.

Compositions and methods using IL-1ra are known in the art. For example,IL-1ra has been delivered as part of a composition with hyaluronic acid,as described in U.S. Pat. No. 6,096,728, Collins et al., issued Aug. 1,2000. However, many such methods and compositions are associated withissues regarding stability and half-life of IL-1ra as well as the amountand rate of IL-1ra provided. Accordingly, improved methods of deliveringIL-1ra are desirable and would be useful in treating conditions andpathologies mediated by the interleukin-1 receptor, including themanagement of inflammation.

SUMMARY

The present technology provides methods for generating solutions rich ininterleukin-1 receptor antagonist and for administering such solutionsto the site of inflammation in a human or animal subject. Methods forgenerating such solutions include incubating adipose tissue withpolyacrylamide beads. A solution rich in interleukin-1 receptorantagonist is then separated from the polyacrylamide beads. The adiposetissue may be obtained from the subject.

Methods of treating a condition mediated by the interleukin-1 receptorin a human or animal subject, such as inflammation, includeco-administering a solution rich in interleukin-1 receptor antagonistand fibrinogen. In various embodiments, such methods further compriseadministration of thrombin and calcium chloride to the subject. The siteof inflammation may be associated, for example, with arthritis, e.g.,osteoarthritis. Preferably, the solution of IL-1ra is autologous.

BRIEF DESCRIPTION OF THE DRAWINGS

The present technology will become more fully understood from thedetailed description and the accompanying drawings, wherein:

FIG. 1 is a diagrammatic illustration of a first method to produce asolution of IL-1ra according to an embodiment of the present technology;

FIG. 2 is a partial cross-sectional view of a representative device usedfor isolating a liquid volume comprising white blood cells and plateletsaccording to one embodiment of the present technology;

FIGS. 3A and 3B are cross-sectional views of a representative device forincubating a volume of white blood cells and platelets withpolyacrylamide beads according to one embodiment of the presenttechnology;

FIG. 4 is blood component isolation device which may be used in methodsof the present technology;

FIG. 5 is a side view of the blood component isolation device of FIG. 4,illustrating an interior portion of a main chamber of the device;

FIG. 6 is a diagrammatic illustration of a method for delivering IL-1raaccording to an embodiment of the present technology; and

FIG. 7 is a partial cross-sectional view of a representative device fordelivering IL-1ra according to one embodiment of the present technology.

It should be noted that the figures set forth herein are intended toexemplify the general characteristics of materials and methods amongthose of the present technology, for the purpose of the description ofcertain embodiments. These figures may not precisely reflect thecharacteristics of any given embodiment, and are not necessarilyintended to define or limit specific embodiments within the scope ofthis technology.

DETAILED DESCRIPTION

The description of the following technology is merely exemplary innature of the subject matter, manufacture and use of one or moreinventions, and is not intended to limit the scope, application, or usesof any specific invention claimed in this application or in such otherapplications as may be filed claiming priority to this application, orpatents issuing therefrom. The following definitions and non-limitingguidelines must be considered in reviewing the description of thetechnology set forth herein.

The headings (such as “Introduction” and “Summary”) and sub-headingsused herein are intended only for general organization of topics withinthe present disclosure, and are not intended to limit the disclosure ofthe technology or any aspect thereof. In particular, subject matterdisclosed in the “Introduction” may include novel technology and may notconstitute a recitation of prior art. Subject matter disclosed in the“Summary” is not an exhaustive or complete disclosure of the entirescope of the technology or any embodiments thereof. Classification ordiscussion of a material within a section of this specification ashaving a particular utility is made for convenience, and no inferenceshould be drawn that the material must necessarily or solely function inaccordance with its classification herein when it is used in any givencomposition.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. All references citedin the “Detailed Description” section of this specification are herebyincorporated by reference in their entirety.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the apparatus and systems of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the materials, compositions, devices,and methods of this technology. Similarly, the terms “can” and “may” andtheir variants are intended to be non-limiting, such that recitationthat an embodiment can or may comprise certain elements or features doesnot exclude other embodiments of the present technology that do notcontain those elements or features.

“A” and “an” as used herein indicate “at least one” of the item ispresent; a plurality of such items may be present, when possible.“About” when applied to values indicates that the calculation or themeasurement allows some slight imprecision in the value (with someapproach to exactness in the value; approximately or reasonably close tothe value; nearly). If, for some reason, the imprecision provided by“about” is not otherwise understood in the art with this ordinarymeaning, then “about” as used herein indicates at least variations thatmay arise from ordinary methods of measuring or using such parameters.In addition, disclosure of ranges includes disclosure of all distinctvalues and further divided ranges within the entire range.

The present technology relates to interleukin-1 receptor antagonist(IL-1ra), including methods of generating IL-1ra, compositionscomprising IL-1ra produced by such methods, methods of using IL-1ra,treatment methods comprising IL-1ra, and devices for the generation,isolation, and administration of IL-1ra.

Methods for generating a solution rich in interleukin-1 receptorantagonist can include the following aspects. In some embodiments,methods for generating a solution rich in interleukin-1 receptorantagonist include contacting a liquid volume comprising adipocytes withpolyacrylamide beads and separating the liquid volume from thepolyacrylamide beads and the adipocytes to obtain a solution rich ininterleukin-1 receptor antagonist. Without limiting the mechanism,utility, or function of the present technology, the polyacrylamide beadsappear to serve as an activator of IL-1ra production by adipocytes. Insome respects, contact of the adipocytes with the surface of thepolyacrylamide beads appears to stimulate IL-1ra production andsecretion by adipocytes. There also appears to be a correlation betweenthe amount of IL-1ra produced and the concentration of white bloodcells, where adipose tissue can include white blood cells. Thus, thepresent technology uses adipose tissue and disaggregated adipose tissueto obtain adipocytes, where white blood cells can be present in both theadipose tissue and the adipocytes obtained from adipose tissue.

The methods can further include the following aspects. The liquid volumeof adipocytes can be part of isolated adipose tissue; where, forexample, the adipose tissue may include other cell types. Contacting ofthe adipocytes and polyacrylamide beads may include incubating theliquid volume of adipocytes with the polyacrylamide beads for timesranging from about 30 seconds to about 24 hours. The contacting may alsoinclude contacting a liquid volume comprising white blood cells with thepolyacrylamide beads, in addition to contacting the liquid volume ofadipocytes with the polyacrylamide beads. The liquid volume of whiteblood cells can be whole blood, platelet rich plasma, or whole blood andplatelet rich plasma. White blood cells can also be obtained from bonemarrow. In some embodiments, separation to obtain the solution rich ininterleukin-1 receptor antagonist comprises centrifuging the liquidvolume of adipocytes and polyacrylamide beads to obtain a supernatantcomprising the solution rich in interleukin-1 receptor antagonist. Theresulting solution rich in interleukin-1 receptor antagonist can includefrom about 30,000 pg/mL to about 110,000 pg/mL interleukin-1 receptorantagonist.

In some embodiments, methods are provided for generating a solution richin interleukin-1 receptor antagonist that is useful for treating aninflammatory disorder in a patient. These methods include obtainingadipose tissue from the patient and loading the adipose tissue into aconcentrator assembly, where the concentrator assembly includespolyacrylamide beads. The mixture of polyacrylamide beads and adiposetissue is incubated to form a solution of interleukin-1 receptorantagonist. The concentrator assembly is then rotated at centrifugalspeeds to separate the interleukin-1 receptor antagonist from thepolyacrylamide beads and adipose tissue to obtain the solution rich ininterleukin-1 receptor antagonist. Loading of the concentrator assemblymay include incubating the adipose tissue with the polyacrylamide beadsfor a time of from about 30 seconds to about 24 hours. The loading mayfurther include loading a liquid volume comprising white blood cellsinto the concentrator assembly. The liquid volume of white blood cellscan be in the form of whole blood, platelet rich plasma, or acombination of whole blood and platelet rich plasma.

The present technology also includes methods of treating one or moresites of inflammation in a patient. Such methods include contacting aliquid volume comprising adipocytes with polyacrylamide beads. Theliquid volume is then separated from the polyacrylamide beads and theadipocytes to provide a solution rich in interleukin-1 receptorantagonist. The solution rich in interleukin-1 receptor antagonist isadministered one or more sites of inflammation in the patient. Theadipose tissue used may be derived from the patient; i.e., autologous.The method can be applied to treat inflammation associated withosteoarthritis.

In some embodiments, the present methods include administeringfibrinogen, thrombin, and calcium to the site of inflammation inaddition to administering the solution rich in interleukin-1 receptorantagonist. For example, methods can include co-administering (i) afirst solution comprising the interleukin-1 receptor antagonist andfibrinogen, and (ii) a second solution comprising thrombin and calcium.

Thrombin used in the present methods may be made by a process thatincludes loading whole blood or plasma and a calcium solution into ablood isolation device. The whole blood or plasma is heated for at leastabout 20 minutes, at a temperature of at least about 20° C. Thrombin isisolated by centrifuging the heated whole blood or plasma. The wholeblood or plasma may be obtained from the patient.

Also provided are methods of treating an inflammatory disorder in apatient. Such methods include obtaining adipose tissue from the patientand loading the adipose tissue into a concentrator assembly, where theassembly includes polyacrylamide beads. The mixture of beads and adiposetissue is incubated to form a solution of interleukin-1 receptorantagonist. The concentrator assembly is then rotated at centrifugalspeeds to separate the interleukin-1 receptor antagonist from thepolyacrylamide beads and obtain a solution rich in interleukin-1receptor antagonist. Whole blood is obtained from the patient andloaded, along with a calcium solution, into a blood isolation device.The whole blood is heated for at least about 20 minutes at a temperatureof at least about 20° C. The heated whole blood centrifuged to obtain aclotting fraction. The solution rich in interleukin-1 receptorantagonist and the clotting fraction are then administered to the siteof the inflammation in the patient.

Treatment methods can further include the following aspects. Loading ofthe adipose tissue into the concentrator assembly, where the assemblyincludes polyacrylamide beads, can include loading a liquid volumecomprising white blood cells with the adipose tissue into and incubatingthe mixture of beads, adipose tissue, and white blood cells to form asolution of interleukin-1 receptor antagonist. The liquid volume ofwhite blood cells can be whole blood, platelet rich plasma, or wholeblood and platelet rich plasma. Fibrinogen may also be administered tothe site of the inflammation in the patient along with the solution richin interleukin-1 receptor antagonist and the clotting fraction. Themethods can be used to treat inflammation due at least in part toosteoarthritis.

Referring now to FIG. 1, a diagrammatic illustration is shown of amethod 100 for generating a solution rich in IL-1ra. Adipose tissue isisolated from a patient at step 110. This adipose tissue may be useddirectly in step 130, or may be processed to provide adipocytes in step120.

Adipose tissue refers to any fat tissue, either white or brown adiposetissue, which may be derived from subcutaneous, omental/visceral,mammary, gonadal, or other adipose tissue sites. In some embodiments,adipose tissue is derived from human subcutaneous fat isolated bysuction assisted lipectomy or liposuction. Adipocytes may be isolatedand/or freed from the adipose tissue and/or tissue portions using anysuitable method, including methods known in the art such as mechanicaland breakdown centrifugation. Adipocytes can also be isolated usingenzymatic digestion. For example, adipocytes can be isolated fromlipoaspirate, treated by sonication and/or enzymatic digestion, andenriched by centrifugation. Adipocytes isolated from adipose tissue maybe washed and pelleted.

Methods for isolating adipose tissue and adipocytes can include thefollowing aspects. About 50 cc of adipose tissue is collected bysuction-assisted tumescent liposuction inside a specialized collectioncontainer attached to suction hoses and to a liposuction cannula. Thecollection container can have a gauze-type grid filter that allows thetumescent fluid to pass through and retains the solid adipose tissue.After collecting the adipose tissue, the collection container is removedfrom the suction device and reattached to a centrifugation device. Thefilter unit may further contain a filter having approximately a 100micrometer pore size. Once the collection container containing theadipose tissue is attached to the centrifugation device, the tissue issonicated. After sonication, the entire apparatus is inserted into acentrifuge bucket and centrifuged at, for example, 300×g for 5 minutes.After centrifugation, the collection container together with the filterunit is detached and can be discarded. The pellet containing theadipocytes can then be resuspended in biocompatible solutions, such asautologous plasma, plasma concentrate and platelet rich plasma.

Adipose tissue may also be treated with digestive enzymes and withchelating agents that weaken the connections between neighboring cells,making it possible to disperse the tissue into a suspension ofindividual cells, including adipocytes, without appreciable cellbreakage. Following disaggregation, the adipocytes may be isolated fromthe suspension of cells and disaggregated tissue.

Various methods and devices for isolating and/or fractionating adiposetissue include those as described by U.S. Pat. Nos. 7,374,678 and7,179,391 to Leach et al. and U.S. Pub. Nos. 2009/0014391, 2008/0283474,and 2007/0208321 to Leach et al. A device, such as the GPS™ PlateletConcentrate System (Biomet, Warsaw, Ind.), may be used to isolateadipocytes. These methods may include obtaining adipocytes by performinglipoaspiration on the patient to obtain adipose tissue, enzymaticallydigesting the adipose tissue, and separating and/or washing theadipocytes using these devices.

As shown at step 130 of FIG. 1, the adipose tissue and/or adipocytes arecontacted with polyacrylamide beads. In some embodiments, the adiposetissue and/or adipocytes are incubated with the polyacrylamide beads fora time effective to remove a portion of the liquid in the liquid volumeof white blood cells and platelets. The incubation may be carried outover a period from about 30 seconds to about 72 hours and may be carriedout at a temperature from about 20° C. to about 41° C. For example, theincubation may be from about one minute to about 48 hours, from about 5minutes to about 12 hours, or from about 10 minutes to about 6 hours. Insome embodiments, the incubation is conducted at about 37° C. In someembodiments the adipose tissue and/or adipocytes are not incubated, butis contacted with the polyacrylamide beads for only so long as necessaryto perform subsequent processing. The contacting may occur at ambientconditions, e.g., at a temperature of about 20-25° C.

Polyacrylamide beads used in step 130 can be formed by polymerizingacrylamide monomer using controlled and standardized protocols as knownin the art to produce relatively uniform beads formed of polyacrylamidegel. In general, polyacrylamide is formed by polymerizing acrylamidewith a suitable bifunctional crosslinking agent, most commonlyN,N-methylenebisacrylamide (bisacrylamide). Gel polymerization isusually initiated with ammonium persulfate and the reaction rate isaccelerated by the addition of a catalyst, such asN,N,N′,N′-tetramethylethylenediamine (TEMED). In various embodiments,polyacrylamide beads comprise 0.5 micromole of carboxyl groups permilliliter of beads, imparting a slight anionic character (negativecharge). The beads are also typically resistant to changes in pH, andare stable in many aqueous and organic solutions. By adjusting the totalacrylamide concentration, the polyacrylamide gel can be formed in a widerange of pore sizes. Moreover, the polyacrylamide beads can be formed inmany sizes and can have relatively uniform size distributions. Bead sizemay range from several micrometers in diameter to several millimeters indiameter. For example, various types of Bio-Gel™ P polyacrylamide gelbeads (Bio-Rad Laboratories, Hercules, Calif., USA) have particle sizesranging from less than about 45 μm up to about 180 μm. Polyacrylamidebeads are also available from SNF Floerger (Riceboro, Ga., USA), PierceBiotechnology, Inc. (Rockford, Ill., USA), and Polymers, Inc.(Fayetteville, Ark., USA).

Once polymerized, polyacrylamide beads can be dried and stored in apowder-like form. The dry beads are insoluble in water but can swellconsiderably upon being rehydrated. Rehydration returns thepolyacrylamide beads to a gel consistency that can be from about two toabout three times the dry state size. Thus, dry polyacrylamide beads maybe used to absorb a portion of a liquid volume, including solutessmaller than the bead pore size, and can serve to concentrate the IL-1raproduced by the adipocytes and/or adipose tissue. For example, combiningdry polyacrylamide beads with the adipocytes and/or adipose tissue instep 130 activates production of IL-1ra and also reduces the totalliquid volume as the dry beads rehydrate and swell.

Without limiting the mechanism, utility or function of the presenttechnology, the polyacrylamide beads may serve as an activator of IL-1raproduction by adipocytes. Therefore, in the case of dry polyacrylamidebeads, not only is liquid being absorbed from the volume of adipocytes,thereby concentrating the IL-1ra formed, but the beads further serve asa surface to stimulate IL-1ra production by the adipocytes. It appearsthat the increase in the amount of IL-1ra is not due to simplyincreasing the concentration by reducing the volume of the sample, butis due to activation of the adipocytes by the polyacrylamide beads toincrease production and/or release of IL-1ra.

In some embodiments, a liquid volume comprising white blood cells, suchas platelet-rich plasma and/or whole blood, may also be added to thepolyacrylamide beads and adipose tissue and/or adipocytes in order togenerate IL-1ra. Blood can be centrifuged to isolate platelet-richplasma (PRP) containing white blood cells and platelets, which may belocated in the buffy coat layer following sedimentation. One example ofa device that may be used for isolating platelet-rich plasma at step 120is shown in FIG. 2.

In this regard, the device 200 shown in FIG. 2 includes a container 205,such as a tube, that is placed in a centrifuge after being filled withblood. The container 205 includes a buoy system having an isolator 210and a buoy 215. The buoy 215 has a selected density which is tuned toreach a selected equilibrium position upon centrifugation; this positionlies between a more dense blood fraction and a less dense bloodfraction. During centrifugation, the buoy 215 separates the blood withinthe container 205 into at least two fractions, without substantiallycommingling the fractions, by sedimenting to a position between the twofractions. In this regard, the isolator 210 and the buoy 215 define alayer comprising platelet-rich plasma 220, while less denseplatelet-poor plasma 225 generally fractionates above the isolator 210,and more dense red blood cells 230 generally fractionate below the buoy215. Following centrifugation, a syringe or tube may then beinterconnected with a portion of the buoy system to extract theplatelet-rich plasma, containing white blood cells. In variousembodiments, such devices may be used to generate platelet-rich plasmathat includes a platelet concentration up to about 8-fold higher thanwhole blood and a white blood cell concentration up to about 5-foldhigher than whole blood. The platelet rich plasma may comprise fromabout 80% to about 90% of the white blood cells present in the wholeblood. Such devices that are commercially available include the GPS® IIPlatelet Concentrate System, from Biomet Biologics, LLC (Warsaw, Ind.,USA) and GPS® III Platelet Separation System, from Biomet Biologics, LLC(Warsaw, Ind., USA).

Devices that may be used to isolate platelet-rich plasma at step 120 arealso described, for example, in U.S. Pat. No. 6,398,972, Blasetti etal., issued Jun. 4, 2002; U.S. Pat. No. 6,649,072, Brandt et al., issuedNov. 18, 2003; U.S. Pat. No. 6,790,371, Dolocek, issued Sep. 14, 2004;U.S. Pat. No. 7,011,852, Sukavaneshvar et al., issued Mar. 14, 2006;U.S. Application Publication No. 2004/0251217, Leach et al., publishedDec. 16, 2004 (incorporated by reference herein); U.S. ApplicationPublication No. 2005/0109716, Leach et al., published May 26, 2005(incorporated by reference herein); U.S. Application Publication No.2005/0196874, Dorian et al., published Sep. 8, 2005 (incorporated byreference herein); and U.S. Application Publication No. 2006/0175242,Dorian et al., published Aug. 10, 2006 (incorporated by referenceherein).

Other methods may be used to isolate platelet-rich plasma. For example,whole blood can be centrifuged without using a buoy system, whole bloodmay be centrifuged in multiple stages, continuous-flow centrifugationcan be used, and filtration can also be used. In addition, a bloodcomponent including platelet-rich plasma can be produced by separatingplasma from red blood cells using a slow speed centrifugation step toprevent pelleting of the platelets. In other embodiments, the buffy coatfraction formed from centrifuged blood can be separated from remainingplasma and resuspended to form platelet-rich plasma.

In addition to the GPS® Platelet Concentrate and Separation Systems, avariety of other commercially available devices may be used to isolateplatelet-rich plasma at step 120, including the Magellan™ AutologousPlatelet Separator System, commercially available from Medtronic, Inc.(Minneapolis, Minn., USA); SmartPReP™, commercially available fromHarvest Technologies Corporation (Plymouth, Mass., USA); DePuy (Warsaw,Ind., USA); the AutoloGel™ Process, commercially available fromCytomedix, Inc. (Rockville, Md., USA); the GenesisCS System,commercially available from EmCyte Corporation (Fort Myers, Fla., USA);and the PCCS System, commercially available from Biomet 3i, Inc. (PalmBeach Gardens, Fla., USA).

Blood drawn from the patient may be mixed with an anticoagulant.Suitable anticoagulants include heparin, citrate phosphate dextrose(CPD), ethylenediaminetetraacetic acid (EDTA), anticoagulant citratedextrose solution (ACD), and mixtures thereof. The anticoagulant may beplaced in the syringe used for drawing blood from the subject, or may bemixed with the blood after it is drawn.

White blood cells may also be prepared using other methods known in theart. For example, white blood cells may be prepared from whole blood bylysing red blood cells or by centrifugation of whole blood utilizing adensity gradient where the white blood cells sediment to the bottom of acentrifuge tube. An example of density centrifugation includes theFicoll-Paque™ Plus (GE Healthcare Bio-Sciences, Piscataway, N.J., USA).In some cases, a density gradient may be used to further separatemononuclear and polymorphonuclear cells. White blood cells may also beprepared from whole blood using filtration; an example includes theAcelere™ MNC Harvest System (Pall Life Sciences, Ann Arbor, Mich., USA).White blood cells can also be obtained from bone marrow.

Referring again to FIG. 1, following incubation with the polyacrymidebeads, an IL-1ra-rich solution is isolated from the polyacrymide beadsand adipose tissue and/or adipocytes, as indicated at step 140.Isolation may be accomplished by drawing off the liquid volume andleaving the beads. In some cases, the beads may be sedimented bycentrifugation prior to drawing off the IL-1ra-rich solution. Isolationmay also be performed by filtration, where the polyacrylamide beads areretained by a filter and the IL-1ra-rich solution passes through thefilter using centrifugal force or by using vacuum, for example. If theincubation with polyacrylamide beads at step 130 utilizes drypolyacrylamide beads, the liquid volume may be reduced as the beadsswell upon rehydration, thereby concentrating the resulting IL-1ra-richsolution. To maintain the increased concentration, care should be takenin the isolation step 140 so as to avoid compressing the beads ordrawing liquid out from the swollen beads. For example, high centrifugalforce or high vacuum may collapse the beads and/or draw liquid out ofthe internal volume of the beads.

In some cases, the incubation with polyacrylamide beads, as per step130, and the isolation of the resulting IL-1ra-rich solution, as perstep 140, may be performed using a single device. An example of a devicefor incubating adipose tissue and/or adipocytes with polyacrylamidebeads is shown in FIGS. 3A and 3B. In this regard, the device 300 has anupper chamber 305 and a lower chamber 310. The upper chamber 305 has anend wall 315 through which the agitator stem 320 of a gel bead agitator325 extends. The device 300 also has an inlet port 330 that extendsthrough the end wall 315 and into the upper chamber 305. The device 300also includes an outlet port 335 that communicates with a conduit 340.The floor of upper chamber 305 includes a filter 345, the upper surfaceof which supports desiccated concentrating polyacrylamide beads 350.

During use, a fluid 355 containing adipose tissue and/or adipocytes isinjected to the upper chamber 305 via the inlet port 330 and mixed withthe polyacrylamide beads 350. The fluid 355 and polyacrylamide beads 350may be mixed by rotating the agitator stem 320 and the gel bead agitator325, to help mix the fluid 355 and beads 350. The mixed fluid 355 andpolyacrylamide beads 350 are then incubated for the desired time at thedesired temperature. The device 300 is then centrifuged so that liquidpasses to the lower chamber 310 while the polyacrylamide beads 350 areretained by a filter 345, thereby separating the polyacrylamide beads350 from the resulting solution 360 of IL-1ra that collects in the lowerchamber 310. The solution 360 may be removed from the device via outletport 335.

Exemplary devices of FIG. 3 are disclosed in U.S. ApplicationPublication 2006/0175268, Dorian et al., published Aug. 10, 2006; andU.S. Application Publication 2006/0243676, Swift et al., published Nov.2, 2006; both of which are incorporated by reference herein. Such adevice is commercially available as Plasmax™ Plus Plasma Concentrator,from Biomet Biologics, LLC (Warsaw, Ind., USA).

Referring again to FIG. 1, in step 150 the IL-1ra-rich solution isadministered to a human or animal subject (patient). The patientreceiving the IL-1ra-rich solution may be the same patient from whichthe adipose tissue in step 110 is derived. In this case, the methodprovides an autologous preparation of IL-1ra. Administration may beperformed using various means, such as by injection of the IL-1ra-richsolution using a syringe, surgical application, or applicationconcomitant with another surgical procedure. It should be understood,however, that step 150 may comprise any biomedically acceptable processor procedure by which the IL-1ra-rich solution is implanted, injected,or otherwise administered in or in proximity to a site in order tomediate effects related to stimulation of the interleukin-1 receptor,such as inflammation and inflammation due to osteoarthritis. Forexample, for treating inflammation caused by arthritis, an autologousIL-1ra-rich solution may be administered to the patient via injection.Injection may be located at or into the synovial space of an inflamedjoint, or otherwise at or near the joint.

The various preparations of IL-1ra-rich solutions produced by thepresent technology may be sterilized by including a sterile filter toprocess the final isolated IL-1ra product. Similarly, an antibiotic maybe included in the polyacrylamide beads during incubation or added atone or more of the various steps in the methods described herein.

The present technology provides improved methods for preparing solutionsrich in IL-1ra, including autologous IL-1ra-rich concentrated plasmasolutions, which reduce and/or substantially eliminate immunologicalissues that may arise when using non-autologous material or recombinantmaterial. In addition, since the IL-1ra is produced by the patient'scells, natural post-translational modifications, such as glycosylation,are already present. This is not the case with most recombinant proteinssince they are produced in prokaryotic hosts.

Solutions rich in IL-1ra generated by the present technology can becharacterized as having increased concentrations of IL-1ra relative tothe concentration of IL-1ra typically found in whole blood. For example,the present methods and compositions can include about 34,000 pg/mL toabout 108,000 pg/mL of IL-1ra, whereas whole blood can include about 200pg/mL to about 800 pg/mL. It is understood, however, the concentrationspresent in any given solution may vary depending on the initial levelsof components present in the adipose tissue, adipocytes, and/or sourceof white blood cells used in the present methods, and that increases inconcentration are relative to those initial levels. In general, IL-1rais present in the present solutions at concentrations of at least about10,000 pg/ml, at least about 25,000 pg/ml, or at least about 30,000pg/ml and can be up to 108,000 pg/mL or more.

The IL-1ra-rich solutions may be administered to mediate effects of IL-1and attenuate signaling via the interleukin-1 receptor. The IL-1ra-richsolution may be used to block the biologic activity of naturallyoccurring IL-1, including inflammation and cartilage degradationassociated with arthritis, by competitively inhibiting the binding ofIL-1 to the interleukin-1 type receptor, which is expressed in manytissues and organs. For example, bone resorption and tissue damage suchas cartilage degradation as a result of loss of proteoglycans due toIL-1 may be treated by administration of the IL-1ra-rich solution. Inpatients with arthritis, endogenous IL-1ra may not be found in effectiveconcentrations in synovium and synovial fluid to counteract IL-1concentrations in these patients, and hence the present IL-1ra-richsolution may be administered to treat these conditions and these sites.Dosing, administration, and frequency of treatment may be modified basedon established medical practices to achieve effective treatment.

Referring again to FIG. 1, in step 150 the IL-1ra-rich solution isadministered to a human or animal subject (i.e., a patient). The patientreceiving the IL-1ra-rich solution may be the same patient from whichthe adipose tissue in step 110 is derived. In this case, the methodprovides an autologous preparation of IL-1ra. Administration may beperformed using various means, such as by injection of the IL-1ra-richsolution using a syringe, surgical application, or applicationconcomitant with another surgical procedure. It should be understood,however, that step 150 may comprise any biomedically acceptable processor procedure by which the IL-1ra-rich solution is implanted, injected,or otherwise administered into or in proximity to a site in order tomediate effects related to stimulation of the interleukin-1 receptor,such as inflammation. For example, for treating inflammation caused byarthritis, an autologous IL-1ra-rich solution may be administered to thepatient via injection. Injection may be located at or into the synovialspace of an inflamed joint, or otherwise at or near the joint.

The present technology further provides methods for delivering IL-1ra.Such delivery methods provide a solution of IL-1ra and fibrinogen wherethe fibrinogen is activated to form a fibrin matrix that protects andretains the IL-1ra at a treatment site. The fibrin matrix can be formedin situ upon delivery of the IL-1ra.

Fibrinogen can be cross-linked into a three-dimensional matrix byactivation with a clotting agent and calcium. Suitable clotting agentsinclude thrombin (e.g., bovine, recombinant human, pooled human, orautologous), autologous clotting protein, and polyethylene glycol.Calcium may be in the form of a calcium salt, such as calcium chloride.

In some embodiments, the clotting agent comprises an autologous clottingprotein, as a clotting fraction or composition derived from bloodobtained from the patient to be treated. A suitable clotting fractioncan be obtained by a process of: loading whole blood or plasma with acalcium solution (e.g., calcium chloride in ethanol) into a bloodisolation device; heating the whole blood or plasma for at least about20 minutes, at a temperature of at least about 20° C.; and isolating theclotting fraction. The isolating may be performed by centrifuging theheated whole blood or plasma. A suitable isolation device is depicted inFIGS. 4 and 5. Such a device is available as the Clotalyst™ AutologousThrombin Collection System from Biomet Biologics LLC, Warsaw, Ind., USA.

With reference to FIGS. 4 and 5, the blood separation device 700generally includes a body having a cylindrical wall along with a firstend 704 and a second end 706 that define a main chamber 702. At thefirst end 704 is a first port 708, a second port 710, a third port 712,a vent 713, and a filter 714. Each of the first port 708, the secondport 710, the third port 712, and the vent 713 extend through the firstend 704 and permit fluid communication between an exterior of the device700 and the main chamber 702. The first port 708 can be covered with afirst cap 716, the second port 710 can be covered with a second cap 718,and the third port 712 can be covered with a third cap 720. A firstreplacement cap 722 for the first port 708 can be attached to the firstport 708 with a first tether 724. A first cover 726 can be secured tothe first replacement cap 722 when the first replacement cap 722 is notin use. A second replacement cap 728 for the second port 710 can beattached to the second port 710 with a second tether 730. A second cover732 can be secured to the second replacement cap 728 when the secondreplacement cap 128 is not in use.

The first port 708 and the second port 710 each include a stop valve toprevent materials, such as glass beads 740, from exiting the mainchamber 702 through the first and the second ports 708 and 710. Thevalves can be any suitable valve, such as a duck-billed valve.

With particular reference to FIG. 5, the third port 712 includes anelongated tube portion 734 that extends within the main chamber 702. Theelongated portion 734 extends from the first end 704 to a depth withinthe main chamber 702 to permit withdrawal of select materials, such asthrombin and other blood clotting factors, from within the main chamber702. For example and as further described below, where the main chamber702 includes whole blood, reagents (e.g., a calcium solution comprisingcalcium compound dissolved in ethanol or other suitable solvent),anticoagulant, and glass beads, incubation and centrifugation of thismixture forms a clotted mass of about including red blood cells, bloodplasma, and glass beads at the second end 706 of the main chamber 702.On top of the clotted mass, at the side of the clotted mass nearest thefirst end 704, an effluent is formed comprising thrombin and variousother clotting factors. The clotted mass at the second end 706 can bevisually distinguished from the effluent. In order to extract thrombinand the other clotting factors using the elongated tube portion 734, theelongated tube portion 734 extends to a depth within the main chamber702 that is approximately level with the portion of the effluent closestto the clotted mass.

A tip 736 is provided at a distal end of the elongated portion 734. Thetip 736 extends from the elongated portion 734 at about a right angle.The tip includes a recess or notch 737. Two support posts 739 extendradially from the elongated portion 734 approximately at the tip 736 tocontact an interior of the main chamber 702. The support posts 739 biasthe tip 736 against the interior of the main chamber 702 to retain thetip 736 at a constant position in the main chamber 702. While the tip736 contacts the interior of the main chamber 702, the notch 737provides an opening or clearance between the interior wall of the mainchamber 702 and the tip 736 to permit the passage of material throughthe notch 737 and into the tip 736. The tip 736 helps to maximize theamount of materials withdrawn through the elongated portion 734,particularly when the main chamber 702 is tilted to bring additionalmaterials surrounding the tip 736 to the notch 737. The two supportposts 739 and the tip 736 help center the elongated portion 734 in themain chamber 702.

The ports 708, 710, and 712 are sized to cooperate with a suitable fluiddelivery or transport device, such as a syringe. For example, the firstport 708 can be sized to cooperate with a reagent syringe to permitpassage of reagent through the first port 708 and into the main chamber702; the second port 710 can be sized to cooperate with a blood syringeto permit passage of blood through the second port 710 and into the mainchamber 702; and the third port 712 can be sized to cooperate with asyringe to permit withdrawal of blood components, such as thrombin andother clotting factors, from within the main chamber 702.

The filter 714 can be any suitable filter for filtering materials asthey are withdrawn from within the main chamber 702 through the thirdport 712. The filter 714 includes a polyester screen that is mountedatop the first port 708 and the second port 710. The polyester screenincludes openings that are in the range of about 15 microns to about 25microns in size. For example, the openings can be about 17 microns insize. In place of or in addition to, the filter 714, a filter similar tothe filter 714 can be provided in the elongated portion 734 or at thetip 736.

The main chamber 702 further includes an activator, such as glass beads740. The negatively charged surface of the glass beads activatesclotting and the release of blood clotting factors, which form theclotted mass at the second end 706 of the main chamber 702. The glassbeads 740 can be any suitable type of glass beads, such as boro-silicatebeads.

An exemplary procedure for producing a clotting agent using the deviceof FIG. 5 begins by injection of a reagent comprising calcium chlorideand ethanol into the main chamber 702 through the first port 708. Afterthe reagent has been injected, the first port 708 is closed using thefirst replacement cap 722. Blood with anticoagulant is injected into themain chamber 702 through the second port 710. After the blood has beeninjected, the second port 710 is closed using the second replacement cap728. Optionally, the syringes and blood separation device 700 arepre-heated to a temperature of about 25° C.

The contents of the blood component separation device 700 are mixed byrepeatedly inverting the device 700, e.g. about twelve times, so as tocontact the blood with the glass beads. After mixing, the device isincubated The incubation process can be at a temperature and for aduration that will permit the contents of the device 700 to be heated atabout 25° C. for about 15 minutes. Upon completion of the incubationperiod, a clotted mass of red blood cells, blood plasma, and glass beadsforms at the second end 706 of the main chamber 702. After incubation iscomplete, the device 700 is shaken enough to dislodge and break-up anygel that may be present. The device 700 is then placed in a suitablecentrifuge and spun at about 3200 RPM for about 15 minutes to separatethrombin from the remaining blood components. After centrifugation, aneffluent of thrombin and other clotting factors separates from theclotted mass. After centrifugation is complete, the third cap 720 isremoved and a suitable extraction device, such a syringe, is used toremove the effluent of thrombin and other clotting factors from withinthe main chamber 702 by way of the third port 712, the elongated portion734, and the tip 736.

Thus, the delivery method of the present technology may includeadministration of IL-1ra, fibrinogen, thrombin, and calcium to form afibrin matrix at the treatment site. Exogenous fibrinogen may be addedto a solution of IL-1ra, for example such as bovine thrombin, preferablyat 1000 U/mL. Or, the IL-1ra solution may already have an adequateamount of endogenous fibrinogen. In the case where the solution ofIL-1ra and/or fibrinogen or preparation thereof includes ananticoagulant, such as ACD-A (anticoagulant citrate dextrose solution),the addition of calcium (with thrombin) to activate the fibrinogenshould exceed the effective amount of any chelator in the anticoagulant.

The IL-1ra-rich solutions prepared using the present methods can providean increased concentration of endogenous fibrinogen relative to wholeblood when whole blood and/or platelet-rich plasma is further added tothe adipocytes and/or adipose tissue and the polyacrylamide beads. Forexample, output of the above methods employing platelet-rich plasma,adipose tissue, polyacrylamide beads, and the device illustrated in FIG.3 results in a solution rich in both IL-1ra and fibrinogen relative towhole blood. Such a device is commercially available as the Plasmax™Plus Plasma Concentrator, from Biomet Biologics, LLC (Warsaw, Ind., USA)and includes those devices and methods of use described in U.S.Application Publication 2006/0175268, Dorian et al., published Aug. 10,2006; and U.S. Application Publication 2006/0243676, Swift et al.,published Nov. 2, 2006; both of which are incorporated by referenceherein. This IL-1ra-rich and fibrinogen-rich solution may be used totreat the patient from which the original whole blood and adipose tissuewere derived; i.e., autologous treatment.

An IL-1ra-rich and fibrinogen-rich solution, prepared using the abovemethods using whole blood, adipose tissue, and polyacrylamide beads withthe Plasmax™ Plus Plasma Concentrator, provides a solution having abouta 3-fold (3×) increase in fibrinogen concentration relative to wholeblood. The fibrin matrix/clot formed from the 3× higher concentration offibrinogen is more substantial than a fibrin clot made from baselinefibrinogen levels and is more resistant to breakdown and resorption.

Referring to FIG. 6, a diagrammatic illustration for delivering IL-1ra900 is shown. At step 910, a solution of IL-1ra is provided. The IL-1rasolution may be prepared using the methods described in the presentdisclosure. Exogenous fibrinogen is added to the IL-1ra solution in step920. The exogenous fibrinogen may be prepared from a different sourcethan the IL-1ra solution, such as a different patient, or may be bovinein origin. Or, the exogenous fibrinogen may be prepared from differentstarting material than the IL-1ra solution, but still from the samesource or patient. For example, the IL-1ra solution and the exogenousfibrinogen may be prepared from different blood samples taken from thesame patient. Alternatively, as shown in step 930, a solution that isenriched in both IL-1ra and fibrinogen is prepared, for example, byusing polyacrylamide beads and the Plasmax™ device, as described herein.A solution of thrombin and calcium is provided in step 940 and isco-administered with the solution of IL-1ra to a treatment site.Thereafter, as shown in step 950, the fibrin in the combined solutionscross-links in situ, forming a matrix at the treatment site that servesto protect, retain, and slow release of the IL-1ra.

Delivery of IL-1ra may include co-administering a first solution ofIL-1ra and fibrinogen and a second solution of thrombin and calcium to asubject. In such embodiments, the first solution and second solution arekept separate until administered so that the fibrinogen does not form afibrin matrix until after the solutions are mixed and injected into atreatment site. The solutions may be mixed just before delivery to thetreatment site or may be mixed at the treatment site.

Referring to FIG. 7, a dual syringe device 1000 may be employed in amedically appropriate procedure. The dual syringe device 1000 includes afirst barrel 1005 and a second barrel 1010, both of which are connectedto a mixing chamber 1015. A first plunger 1020 is inserted into thefirst barrel 1005 and a second plunger 1025 is inserted into the secondbarrel 1010. The first plunger 1020 and the second plunger 1025 areconnected by a member 1030. The mixing chamber 1015 connects to acannula 1035. The dual syringe device 1000 contains a first solution1040 of IL-1ra and fibrinogen in the first barrel 1005, and a secondsolution 1045 of thrombin and calcium in the second barrel 1010. Duringco-administration, member 1030 is pushed toward the mixing chamber 1015such that the contents of both the first barrel 1005 and the secondbarrel 1010 are pushed into the mixing chamber 1015. The mixed firstsolution 1040 and second solution 1045 travel through the cannula 1035and form a fibrin-matrix 1050 at the treatment site 1055 within apatient's joint 1060.

In the embodiment shown in FIG. 7, the patient's joint 1060 is a kneejoint that includes a femur 1065, a tibia 1070, a fibula 1075, a patella1080, and cartilage 1085. It should be understood, however, that thetreatment site 1055 may be in any joint of a human patient or animal,including shoulders, elbows, wrists, ankles, hips, and the spinalcolumn. In addition, the present methods may be used to treatinflammation in sites within other tissues, such as muscle and tendon.

In some embodiments, the dual syringe device 1000 is used to pierce softtissue of the patient's joint 1060 to administer the mixed firstsolution 1040 and second solution 1045. For example, the cannula 1035may be a hollow needle such as a hypodermic needle. Alternatively, anincision may be made in the patient's joint 1060 to allow entry of thecannula 1035 so that the dual syringe device 800 may enter the treatmentsite 1055.

In some embodiments, which are not shown, the dual syringe device 1000does not have a mixing chamber 1015 and instead includes two cannulas1035, one leading from each barrel to the treatment site 1055. In thiscase, the first solution 1040 and second solution 1045 travel throughthe separate cannulas 1035 and mix together at the treatment site 1055to form a fibrin-matrix 1050, In some embodiments, two separatesingle-barreled syringe devices are employed in place of a dual syringedevice.

The fibrin matrix formed in the present delivery methods can reside atthe treatment site without increasing inflammation. The IL-1ra withinthe fibrin matrix is protected from enzymatic degradation and may bindto the fibrin matrix so that is it slowly released from the matrix overtime. The methods consequently can provide sustained delivery of IL-1raas compared to injection of IL-1ra without the fibrin-matrix carrier.

The present technology can include aspects of U.S. ProvisionalApplication No. 61/031,803 filed Feb. 27, 2008, U.S. ProvisionalApplication No. 61/116,940 filed Nov. 21, 2008, and U.S. ProvisionalApplication No. 61/155,048 filed Feb. 24, 2009 and includes aspects ofPCT/US2009/035541 filed Feb. 27, 2009.

The following specific examples are provided for illustrative purposesof how to make and use the compositions and methods of this technologyand, unless explicitly stated otherwise, are not intended to be arepresentation that given embodiments of this technology have, or havenot, been made or tested.

Example 1

Adipocytes are prepared as follows. Adipose tissue is minced into smallpieces (about 1 cm³) and digested in 2 mg/mL type I collagenase(Worthington Biochemical Corp., Lakewood, N.J.) under intermittentmechanical agitation in a water bath at 37° C. for 180 minutes.Digestion can be neutralized by the addition of medium or ablood-derived solution. The cell suspension is centrifuged (300×g for 7minutes at 25° C.) followed by removal of the supernatant from the cellpellet. The pellet is then resuspended in a compatible solution toprovide a liquid volume comprising adipocytes.

Alternatively, the pellet is suspended with whole blood obtained fromthe subject, and added to a GPS™ Platelet Concentrate System, fromBiomet Biologics, Inc. (Warsaw, Ind.). Following centrifugation, theplatelet-rich plasma layer, which also contains the adipocytes, isextracted from the system.

The adipocytes, optionally including platelet-rich plasma, are thencombined with polyacrylamide beads to stimulate production of IL-1ra.The adipocytes and polyacrylamide beads are separated from the liquidsolution to obtain a solution rich in IL-1ra.

Example 2

A therapeutic composition of IL-1ra is generated from adipocytes.Isolation of human adipocytes is performed by obtaining humansubcutaneous adipose tissue from lipoaspiration/liposuction proceduresand digesting the tissue in collagenase type I solution (WorthingtonBiochemical Corp., Lakewood, N.J.) under gentle agitation for 1 hour at37° C. The dissociated cells are filtered with 500 μm and 250 μm Nitexfilters. The fraction is centrifuged at 300×g for 5 minutes. Thesupernatant is discarded and the cell pellet is resuspended in acompatible liquid solution, such as a blood-derived solution.

The adipocytes are combined with polyacrylamide beads in a device suchas shown in FIGS. 3A and 3B. A fluid 355 containing the adipocytes isinjected to the upper chamber via the inlet port 330 and mixed with thepolyacrylamide beads 350. The fluid 355 and polyacrylamide beads 350 maybe mixed by rotating the agitator stem 320 and the gel bead agitator325, to help mix the fluid 355 and beads 350. The mixed fluid 355 andpolyacrylamide beads 350 are then incubated for the desired time at thedesired temperature. The device 300 is then centrifuged so that liquidpasses to the lower chamber 310 while the polyacrylamide beads 350 areretained by a filter 345, thereby separating the polyacrylamide beads350 from the resulting solution 360 of IL-1ra that collects in the lowerchamber 310. The IL-1ra rich solution 360 may be removed from the devicevia outlet port 335.

Example 3

An IL-1ra-rich solution is created as follows. Adipose tissue isharvested by liposuction from a patient. Whole blood (70 mL)anticoagulated (10%) with ACD-A (Braintree, Mass., USA) is drawn fromthe patient. A portion (10 mL) is reserved for a whole bloodmeasurement. Platelet-rich plasma (PRP) (6 mL) is produced using theGPS® II System (Biomet Biologics, LLC, Warsaw, Ind., USA). Completeblood counts (CBC) are collected for the whole blood and PRP samplesfollowing a validated procedure, as described in Woodell-May J E,Ridderman D N, Swift M J, Higgins J. “Producing Accurate Platelet Countsfor Platelet Rich Plasma: Validation of a Hematology Analyzer andPreparation Techniques for Counting” J Craniofac Surg (2005) September16(5):749-56.

Adipose tissue (about 5 grams) and PRP (about 5 mL) are added to amodified plasma concentration device (Plasmax™, Biomet Biologics LLC,Warsaw, Ind., USA) and incubated with polyacrylamide desiccating beadsin the device for 24 hours at room temperature. Following incubation,the plasma concentration device is centrifuged to separate the solutionrich in IL-1ra.

To analyze baseline IL-1ra levels at time zero, the adipose tissue, PRP,and polyacrylamide samples are activated with 50 μL of thrombin and 10%CaCl₂ (1,000 units/mL). A blood clot is formed and incubated for 30minutes at room temperature. Following incubation, the clot iscentrifuged for 5 minutes at 3,000 rpm. Serum is collected from theclots and retained for ELISA analysis. The IL-1ra rich solution from theplasma concentrator does not require activation by thrombin, and istested directly. All samples are analyzed for IL-1ra using an ELISA kit(IL-1ra Quantikine™ Kit, R&D Systems, Minneapolis, Minn., USA).

Illustrative data is presented as mean±standard deviation. Statisticalsignificance is evaluated with a Student's t-test (a=0.05). Acorrelation analysis is used to compare IL-1ra output and complete bloodcounts (CBC) data.

IL-1ra generated from incubation of adipose tissue and PRP withpolyacrylamide beads provides an increased level of IL-1ra. The baselineserum values of IL-1ra (217±98 pg/mL) are similar to results found inanother study (73±4.8 pg/mL), described in Meijer H, Reinecke J, BeckerC, Tholen G, Wehling P. “The production of anti-inflammatory cytokinesin whole blood by physico-chemical induction” Inflamm. Res. 2003October; 52(10):404-7, even though significant variability betweendonors can exist. The IL-1ra serum levels are statistically higher inoutput of the plasma concentrator following incubation of adipose tissueand PRP with polyacrylamide beads compared to the baseline serum levels.For example, 24-hour incubation of the adipose tissue and PRP withpolyacrylamide beads in the plasma concentration device results in adose of IL-1ra (about 36,000 pg/mL) that is higher than the previouslyreported data from the 24-hour incubation in the ACS device (10,254±165pg/mL).

Example 4

Adipose tissue (120 g) is collected and prepared using GPS®IIIdisposables (Biomet Biologics LLC, Warsaw, Ind., USA). The isolatedadipose tissue is loaded into modified plasma concentration devices(Plasmax®, Biomet Biologics LLC, Warsaw, Ind., USA) and processed. Theoutput is divided into 4 groups; IL-1ra in concentrated plasma with andwithout thrombin activation (1000 U/ml in 1 M CaCl₂), or cell-freeIL-1ra with and without thrombin activation. IL-1ra is measured usingELISA (R&D Systems) over time.

Unclotted samples produce an average of 47.1±2.1 ng over 24 hrs(p=0.34). The cell-free samples produce 33.7±1.5 ng without changingover 24 hrs (p=0.38). Once clotted, the elution of IL-1ra is slowed,with only 28% being eluted after 10 hours. Release in the cell-freesamples is also delayed, but eluted 100% of available IL-1ra after 10hours.

The examples and other embodiments described herein are exemplary andnot intended to be limiting in describing the full scope of compositionsand methods of the present technology. Equivalent changes, modificationsand variations of specific embodiments, materials, compositions andmethods may be made within the scope of the present technology, withsubstantially similar results.

What is claimed is:
 1. A method for generating a solution rich ininterleukin-1 receptor antagonist comprising: (a) contacting isolatedadipose tissue with polyacrylamide beads, wherein the polyacrylamidebeads activate the isolated adipose tissue to produce interleukin-1receptor antagonist to form a solution rich in interleukin-1 receptorantagonist; and (b) separating the solution rich in interleukin-1receptor antagonist from the isolated adipose tissue and thepolyacrylamide beads.
 2. The method for generating a solution rich ininterleukin-1 receptor antagonist according to claim 1, wherein thecontacting comprises incubating the isolated adipose tissue with thepolyacrylamide beads for a time of from about 30 seconds to about 24hours.
 3. The method for generating a solution rich in interleukin-1receptor antagonist according to claim 1, wherein the contacting furthercomprises contacting a liquid volume comprising white blood cells withthe polyacrylamide beads.
 4. The method for generating a solution richin interleukin-1 receptor antagonist according to claim 3, wherein theliquid volume comprising white blood cells is whole blood, platelet richplasma, or whole blood and platelet rich plasma.
 5. The method forgenerating a solution rich in interleukin-1 receptor antagonistaccording to claim 1, wherein the separating comprises centrifuging theisolated adipose tissue and polyacrylamide beads to obtain a supernatantcomprising the solution rich in interleukin-1 receptor antagonist. 6.The method for generating a solution rich in interleukin-1 receptorantagonist according to claim 1, wherein the solution rich ininterleukin-1 receptor antagonist comprises from about 30,000 pg/mL toabout 110,000 pg/mL interleukin-1 receptor antagonist.
 7. A method forgenerating a solution rich in interleukin-1 receptor antagonist fortreating an inflammatory disorder in a patient, the method comprising:(a) obtaining adipose tissue from the patient; (b) loading the adiposetissue into a concentrator assembly including polyacrylamide beads andincubating the mixture of polyacrylamide beads and adipose tissue toform a solution of interleukin-1 receptor antagonist; and (c) rotatingthe concentrator assembly at centrifugal speeds to separate theinterleukin-1 receptor antagonist from the polyacrylamide beads andadipose tissue to obtain the solution rich in interleukin-1 receptorantagonist.
 8. The method for generating a solution rich ininterleukin-1 receptor antagonist according to claim 7, wherein theloading comprises incubating the adipose tissue with the polyacrylamidebeads for a time of from about 30 seconds to about 24 hours.
 9. Themethod for generating a solution rich in interleukin-1 receptorantagonist according to claim 7, wherein the loading further comprisesloading a liquid volume comprising white blood cells into theconcentrator assembly.
 10. The method for generating a solution rich ininterleukin-1 receptor antagonist according to claim 9, wherein theliquid volume comprising white blood cells is whole blood, platelet richplasma, or whole blood and platelet rich plasma.